Comprehensive Expression Analysis of the WRKY Gene Family in Phoebe bournei under Drought and Waterlogging Stresses

In response to biotic and abiotic stresses, the WRKY gene family plays a crucial role in plant growth and development. This study focused on Phoebe bournei and involved genome-wide identification of WRKY gene family members, clarification of their molecular evolutionary characteristics, and comprehensive mapping of their expression profiles under diverse abiotic stress conditions. A total of 60 WRKY gene family members were identified, and their phylogenetic classification revealed three distinct groups. A conserved motif analysis underscored the significant conservation of motif 1 and motif 2 among the majority of PbWRKY proteins, with proteins within the same class sharing analogous gene structures. Furthermore, an examination of cis-acting elements and protein interaction networks revealed several genes implicated in abiotic stress responses in P. bournei. Transcriptomic data were utilized to analyze the expression patterns of WRKY family members under drought and waterlogged conditions, with subsequent validation by quantitative real-time PCR (RT-qPCR) experiments. Notably, PbWRKY55 exhibited significant expression modulation under drought stress; PbWRKY36 responded prominently to waterlogging stress; and PbWRKY18, PbWRKY38, and PbWRKY57 demonstrated altered expression under both drought and waterlogging stresses. This study revealed the PbWRKY candidate genes that potentially play a pivotal role in enhancing abiotic stress resilience in P. bournei. The findings have provided valuable insights and knowledge that can guide further research aimed at understanding and addressing the impacts of abiotic stress within this species.


Introduction
Abiotic stress factors such as drought and waterlogging seriously affect the growth and development process of trees and seriously limit the production and sustainable development of forestry.Drought is one of the important abiotic stress factors that hinder plant growth and development, and it is also one of the most common forms of plant damage.Waterlogging stress results in root hypoxia, which induces a series of physiological changes.Unfortunately, prolonged anaerobic respiration can result in the accumulation of harmful substances in the rhizosphere.With global warming and environmental degradation, these problems are becoming more and more significant.Therefore, improving the drought resistance and waterlogging resistance of forest trees has become the focus of current research on forest tree breeding.
Phoebe bournei (Hemsl.)Yang, an evergreen broad-leaved tree species classified under the genus Phoebe in the Lauraceae family, holds the status of a class II protected plant in China because of its profound ecological, medicinal, and economic value.Renowned for its fine wood with a beautiful texture, robust corrosion resistance, and minimal susceptibility to cracking during drying, this tree species has been widely used in shipbuilding, construction, and wood artistry [1].However, P. bournei faces challenges such as drought, waterlogging, and salinization, leading to a decline in population size and scattered distribution patterns [2].Despite its environmental relevance, the intricate response mechanism of P. bournei to various stressors remains relatively unexplored.
The WRKY gene family is an important transcriptional regulator that plays an important regulatory role in plant resistance to biotic and abiotic stresses.The structural domain of WRKYGQK consists of approximately sixty strongly conserved amino acid residues, with an invariant heptapeptide amino acid sequence at the end of the N-terminus.There are two zinc-finger motifs at the C-terminus, C 2 H 2 (C-X 4-5 -C-X 22-23 -H-X-H) or C 2 HC (C-X 7 -C-X 23 -H-X-C) [3].Studies have shown that an amino acid in WRKYGQK is substituted, resulting in a change in its sequence, including WRKYGKK, WRKYGEK, WRKYGRK, and WRKYGQK.Additionally, the amino acids RR, SK, KR, VK, IK, RM, RI, or KK of the WRKY structural domain often replace the original "RK" [4].The WRKY transcription factor (TF) can specifically bind to the W-box (TTGACC/T) in the promoter of target genes to activate or repress transcription to regulate the expression of downstream genes [5].The WRKY family is classified according to the number of WRKY structural domains and the type of zinc-finger motif.Group I contains two WRKY conserved domains, and the zinc-finger structure is C 2 H 2 .Groups II and III both contain only one WRKY conserved domain.The Group II zinc-finger structure is the same as that of Group I, which is C 2 H 2 , while the Group III zinc-finger structure is C 2 HC.Additionally, according to conserved structural motifs other than WRKYGQK, Group II can be divided into IIa, IIb, IIc, IId, and IIe [6,7].
Many studies have reported that WRKY TFs regulate gene expression and play a variety of biological functions, especially in abiotic stress responses [8,9].The overexpression of an Arabidopsis thaliana WRKY TF (AtWRKY30) significantly enhances the ability of wheat to resist heat and drought [10], and AtWRKY53 negatively regulates drought resistance in A. thaliana by mediating stomatal movement [11].AtWRKY57 can improve A. thaliana drought resistance by increasing the level of abscisic acid ABA [12].In rice, OsWRKY11 plays a significant role in rice resistance to high temperature and drought, improving the stress resistance of plants [13].Similarly, the expression of OsWRKY45 increases significantly under ABA, NaCl, and polyethylene glycol PEG treatments, and the drought resistance of A. thaliana is enhanced [14].Phyllostachys edulis, PheWRKY72-2 can enhance drought tolerance in transgenic A. thaliana by regulating stomatal closure [15].In Camellia japonica, CjWRKY21 is overexpressed during low-temperature pressure, and CjWRKY22 is overexpressed during drought damage [16].
Although the WRKY gene family has been thoroughly researched in many plants, such as A. thaliana [17], Zea mays [18], Oryza sativa [13], and other plants, there has been no systematic analysis of the WRKY gene family in P. bournei.In particular, little is known about the response of P. bournei to abiotic stresses at the molecular level.In this study, members of the WRKY gene family were identified, and the phylogenetic relationship, gene structure, motif composition, cis-acting elements, exon-intron structure, evolutionary pattern, protein structure and interaction regulatory network, and gene expression patterns under waterlogging and drought stress were systematically analyzed.Additionally, the expression level of PbWRKY with different expression patterns was verified by a quantitative real-time PCR (RT-qPCR) analysis.This study provides a scientific basis for the in-depth study of the biological function of PbWRKY genes and their response mechanism in abiotic stress and also provides theoretical guidance for breeding P. bournei varieties with excellent resistance.

The WRKY Gene Family in P. bournei
The conserved domain of the P. bournei WRKY was determined by the A. thaliana WRKY model.Finally, 60 WRKY were obtained and named PbWRKY1-PbWRKY60 (Table 1).
The physical and chemical properties (including amino acids, molecular weight, isoelectric point, and subcellular location) of the WRKY protein were analyzed by ExPaSy.Among the 60 WRKY proteins, there was a significant difference in the number of amino acids, with a minimum number of amino acids (104) and a maximum number of amino acids (883), a molecular weight range of 12.64 to 96.25 kDa, and an isoelectric point range of 4.80 to 10.80.Subcellular localization predicted that many PbWRKY proteins belong to nuclear proteins, while only PbWRKY52 may belong to chloroplasts or nuclei.

PbWRKY Protein Phylogenetic Analysis
There were three groups of PbWRKY proteins (Figure 1): Group I in yellow, which contains 13 PbWRKYs; Group IIa in red, which contains 3 PbWRKYs; Group IIb in green, which contains five PbWRKYs; Group IIc in blue, which contains 16 PbWRKYs; Group IId in orange, which contains five PbWRKYs; Group IIe in purple, which contains 11 PbWRKYs; and the number of members was lowest in Group IIa and highest in Group IIc.The proportion of PbWRKY genes in each group was different from that of the AtWRKY genes in A. thaliana.Group I had the highest number of WRKY proteins in A. thaliana, but Group II had the most predominant WRKY proteins in P. bournei.

PbWRKY Protein Phylogenetic Analysis
There were three groups of PbWRKY proteins (Figure 1): Group I in yellow, which contains 13 PbWRKYs; Group IIa in red, which contains 3 PbWRKYs; Group IIb in green, which contains five PbWRKYs; Group IIc in blue, which contains 16 PbWRKYs; Group IId in orange, which contains five PbWRKYs; Group IIe in purple, which contains 11 PbWRKYs; and the number of members was lowest in Group IIa and highest in Group IIc.The proportion of PbWRKY genes in each group was different from that of the AtWRKY genes in A. thaliana.Group I had the highest number of WRKY proteins in A. thaliana, but Group II had the most predominant WRKY proteins in P. bournei.

PbWRKY Multiple Sequence Alignment
PbWRKY domains and conserved motifs spanning 60 amino acids were investigated using multiple sequence alignment (Figure 2).The investigation of the PbWRKY subfamily conserved domains revealed that the members of the subfamily contained highly conserved WRKY domains, individual members with WRKYGQK heptapeptide domain variants, and zinc-finger structure variants.Group I contained two WRKY conserved domains.They were classified as I-N and I-C according to their position in the sequence.Group I-N PbWRKYs contained a WRKYGQK domain and a CX4CX22HXH zinc-finger structure.Group I-C PbWRKYs contained a WRKYGQK domain and a CX4CX23HXH zinc-finger structure.Relative to Groups I, II, and III, only one conserved domain exists.

PbWRKY Multiple Sequence Alignment
PbWRKY domains and conserved motifs spanning 60 amino acids were investigated using multiple sequence alignment (Figure 2).The investigation of the PbWRKY subfamily conserved domains revealed that the members of the subfamily contained highly conserved WRKY domains, individual members with WRKYGQK heptapeptide domain variants, and zinc-finger structure variants.Group I contained two WRKY conserved domains.They were classified as I-N and I-C according to their position in the sequence.Group I-N Pb-WRKYs contained a WRKYGQK domain and a CX4CX22HXH zinc-finger structure.Group I-C PbWRKYs contained a WRKYGQK domain and a CX4CX23HXH zinc-finger structure.Relative to Groups I, II, and III, only one conserved domain exists.Group II was further divided into five distinct groups (Group IIa-Group IIe) based on the amino acid sequence differences.Groups IIa, IIb, IId, and IIe were similar, containing a WRKYGQK domain and a CX5CX23HXH zinc-finger structure.A WRKYGQK domain and a CX4CX23HXH zinc-finger structure were found in Group IIc PbWRKYs.A WRKYGQK domain and a CX7CX23HXC zinc-finger structure were found in Group III PbWRKYs.

PbWRKY Gene Structure and Motif Composition
Sixty PbWRKY protein sequences contained 20 motifs: the number of motifs in PbWRKYs ranged from one (PbWRKY1) to nine (PbWRKY17, PbWRKY24, PbWRKY28, and PbWRKY57) (Figure 3B).Motifs 1, 2, and 20 were observed in Group III of PbWRKY proteins, but motifs 1, 2, 4, 8, 11, 16, and 18 were identified in Group IId, whereas motifs 1, 2, 10, and 12 were observed in Group IIa.PbWRKYs all had motifs 1 and 2, while motifs 3, 9, 13, and 17 were only identified in Group I, and motifs 5, 6, and 18 were only identified in Group IId.Motifs 15 and 19 were unique to Groups IIc and IIe.Most branches containing motifs 4 and 16 were present in Group I, but PbWRKY10, which contained motif 16, was

PbWRKY Gene Structure and Motif Composition
Sixty PbWRKY protein sequences contained 20 motifs: the number of motifs in Pb-WRKYs ranged from one (PbWRKY1) to nine (PbWRKY17, PbWRKY24, PbWRKY28, and PbWRKY57) (Figure 3B).Motifs 1, 2, and 20 were observed in Group III of PbWRKY proteins, but motifs 1, 2, 4, 8, 11, 16, and 18 were identified in Group IId, whereas motifs 1, 2, 10, and 12 were observed in Group IIa.PbWRKYs all had motifs 1 and 2, while motifs 3, 9, 13, and 17 were only identified in Group I, and motifs 5, 6, and 18 were only identified in Group IId.Motifs 15 and 19 were unique to Groups IIc and IIe.Most branches containing motifs 4 and 16 were present in Group I, but PbWRKY10, which contained motif 16, was located in Group IIc.The exon/intron structure and number of exons were analyzed to detect features of the evolutionary events in the PbWRKY genes family in P. bournei (Figure 3C).The PbWRKY gene contained two to eight exons, with PbWRKY1 and PbWRKY52 having eight exons (n = 8).PbWRKYs had similar exons and introns in the same group, such as in Groups IIa and IId, which had a low number of exons and a conserved structure.PbWRKY Group III contained two to three exons, with approximately 86% (6/7) containing three exons.Group IIe contained three to seven exons, with approximately 64% (7/11) containing three exons, and Group IIb contained four to seven exons, with approximately 60% (3/5) containing five exons.In conclusion, almost all PbWRKYs of the same group or subgroup had very similar motif compositions and gene structures.This indicated that these PbWRKY proteins may have similar functions, further validating the phylogenetic relationship of PbWRKYs.

Ka and Ks of the PbWRKY Gene Family
The origin and evolution of the PbWRKYs were further identified by calculating the Ka and Ks values for homologous gene pairs.We identified a total of 21 homologous gene pairs in the P. bournei genome (Table 2).The Ks values ranged from 0.07 to 3.14 for P. bournei homologous pairs, indicating that the duplication event occurred approximately 5.4−241.94 million years ago, whereas only six homologous pairs were identified in P. The exon/intron structure and number of exons were analyzed to detect features of the evolutionary events in the PbWRKY genes family in P. bournei (Figure 3C).The PbWRKY gene contained two to eight exons, with PbWRKY1 and PbWRKY52 having eight exons (n = 8).PbWRKYs had similar exons and introns in the same group, such as in Groups IIa and IId, which had a low number of exons and a conserved structure.PbWRKY Group III contained two to three exons, with approximately 86% (6/7) containing three exons.Group IIe contained three to seven exons, with approximately 64% (7/11) containing three exons, and Group IIb contained four to seven exons, with approximately 60% (3/5) containing five exons.In conclusion, almost all PbWRKYs of the same group or subgroup had very similar motif compositions and gene structures.This indicated that these PbWRKY proteins may have similar functions, further validating the phylogenetic relationship of PbWRKYs.

Ka and Ks of the PbWRKY Gene Family
The origin and evolution of the PbWRKYs were further identified by calculating the Ka and Ks values for homologous gene pairs.We identified a total of 21 homologous gene pairs in the P. bournei genome (Table 2).The Ks values ranged from 0.07 to 3.14 for P. bournei homologous pairs, indicating that the duplication event occurred approximately 5.4-241.94 million years ago, whereas only six homologous pairs were identified in P. bournei and A. thaliana (Table 3).The Ks values for P. bournei and A. thaliana homologous gene pairs ranged from 1.82 to 4.03, suggesting that the replication event occurred between 140.37 and 309.77 million years ago.To understand the evolutionary selection pressure on different PbWRKY gene pairs, we also calculated the Ka/Ks ratio.Ka/Ks indicates a positive or diversifying selection when Ka or Ks is >1 and a neutral selection when Ka or Ks = 1.The results showed that the Ka/Ks ratios of the 21 PbWRKY homologous gene pairs were all <0.5 (Table 2), indicating that the WRKY gene family of P. bournei had undergone strong purifying selection during evolution, eliminating most of the deleterious mutations and resulting in a high level of protein conservation.Six P. bournei and A. thaliana homologous gene pairs (except PbWRKY37-AtWRKY34) had Ka/Ks ratios that were all <0.4 (Table 3), indicating that the WRKY gene families of P. bournei and A. thaliana have undergone mainly strong negative selection during evolution.

Analysis of Cis-Acting Elements in the Promoter Region of PbWRKYs
The cis-acting elements of the promoter sequence of PbWRKYs were obtained using the PlantCARE database to understand and speculate their possible response mechanisms under various stresses.The 56 cis-elements identified could be classified into four categories according to their functions, including 26 light-responsive elements, 12 hormone-related elements, 12 development elements, and six abiotic stress-responsive elements (Figure 4).A total of 111 G-boxes were the most abundant among all cis-elements, in addition to 48 Box 4 elements and 39 GT1 motifs, suggesting that the expression of PbWRKYs was mainly induced by light.Additionally, 100 ABA response elements (ABREs), 52 CGTCA motifs, and 27 TCA elements (salicylic acid response elements) were identified, indicating that PbWRKYs play a large part in the hormone response process.Significantly, many stress response elements were identified in the PbWRKYs, including 84 ARE elements (anaerobic elements), thirty MBS elements (drought response elements), and sixteen TC-rich repeat elements (defense and stress response elements).
teen TC-rich repeat elements (defense and stress response elements).
As shown in Figure 5, fifty-three of the sixty PbWRKY genes possessed different cisacting elements, while seven other PbWRKYs were not detected due to short sequences upstream of the translation initiation site.These five cis-acting elements had a different distribution across the different PbWRKY gene families.Most of the PbWRKYs contained cis-elements essential for anaerobic induction (83.02%), followed by cis-elements associated with drought stress (49.06%).There were fewer development-related motifs.The CAT-box only had 29 motifs, and the AuxRR-core, circadian, and GON4 motifs had nearly eight motifs, while the others had nearly two or three motifs.From the results, members of the same group or subgroup shared similar potential functions (Figure 5).For example, none of the PbWRKY genes in Group IId had cis-elements related to anoxia-specific induction and low-temperature responses, indicating that this subgroup may not function in this area.Similarly, none of the members of Group III contained enhancer-like elements related to anoxia-specific induction, and therefore, members of this group may not respond to anoxic stress (waterlogging stress, etc.).Members of Group IIb lacked the MYB binding site elements involved in drought induction, implying that members of this group may not respond to drought stress.This indicates that PbWRKYs are responsive to a variety of abiotic stresses.As shown in Figure 5, fifty-three of the sixty PbWRKY genes possessed different cisacting elements, while seven other PbWRKYs were not detected due to short sequences upstream of the translation initiation site.These five cis-acting elements had a different distribution across the different PbWRKY gene families.Most of the PbWRKYs contained cis-elements essential for anaerobic induction (83.02%), followed by cis-elements associated with drought stress (49.06%).There were fewer development-related motifs.The CAT-box only had 29 motifs, and the AuxRR-core, circadian, and GON4 motifs had nearly eight motifs, while the others had nearly two or three motifs.From the results, members of the same group or subgroup shared similar potential functions (Figure 5).For example, none of the PbWRKY genes in Group IId had cis-elements related to anoxia-specific induction and low-temperature responses, indicating that this subgroup may not function in this area.Similarly, none of the members of Group III contained enhancer-like elements related to anoxia-specific induction, and therefore, members of this group may not respond to anoxic stress (waterlogging stress, etc.).Members of Group IIb lacked the MYB binding site elements involved in drought induction, implying that members of this group may

Protein Tertiary Structure Prediction
To further validate the results for the different groupings of PbWRKYs, the protein tertiary structure of PbWRKYs was predicted using the SWISS-MODE online software (https://swissmodel.expasy.org/(accessed on 25 June 2024)).Each final structure was the most predicted structure in its respective grouping (Figure 6).The results indicated that the tertiary structure of the proteins in each group or subgroup differed significantly.The members of the different subgroups all had similar proportions to those of the corresponding structures, representing a group or subgroup with similar protein structures that may perform similar functions.PbWRKY36 in Group I had 90% homology (Seq Identity) to AtWRKY4, and the value of the qualitative model energy analysis (QMEAN) was 0.59; PbWRKY46 in Group IIa had 80.30% homology with AtWRKY18, and the value of QMEAN was 0.61; PbWRKY38 in Group IIb had 58.18% homology with AtWRKY4, and the value of QMEAN was 0.49; PbWRKY20 in Group IIc had 66.20% homology with AtWRKY4, and the value of QMEAN was 0.58; PbWRKY17 in Group IId had 55.71% homology with At-WRKY1, and the value of QMEAN was 0.67; PbWRKY45 in Group IIe had 62.34% homology with RRS1, and the value of QMEAN was 0.66; PbWRKY41 in Group III had 57.81% homology with OsWRKY45, and the value of QMEAN was 0.55.PbWRKY had the highest homology with AtWRKY4 and was most likely a homologous protein.

Protein Tertiary Structure Prediction
To further validate the results for the different groupings of PbWRKYs, the protein tertiary structure of PbWRKYs was predicted using the SWISS-MODE online software (https://swissmodel.expasy.org/(accessed on 25 June 2024)).Each final structure was the most predicted structure in its respective grouping (Figure 6).The results indicated that the tertiary structure of the proteins in each group or subgroup differed significantly.The members of the different subgroups all had similar proportions to those of the corresponding structures, representing a group or subgroup with similar protein structures that may perform similar functions.PbWRKY36 in Group I had 90% homology (Seq Identity) to AtWRKY4, and the value of the qualitative model energy analysis (QMEAN) was 0.59; PbWRKY46 in Group IIa had 80.30% homology with AtWRKY18, and the value of QMEAN was 0.61; PbWRKY38 in Group IIb had 58.18% homology with AtWRKY4, and the value of QMEAN was 0.49; PbWRKY20 in Group IIc had 66.20% homology with AtWRKY4, and the value of QMEAN was 0.58; PbWRKY17 in Group IId had 55.71% homology with AtWRKY1, and the value of QMEAN was 0.67; PbWRKY45 in Group IIe had 62.34% homology with RRS1, and the value of QMEAN was 0.66; PbWRKY41 in Group III had 57.81% homology with OsWRKY45, and the value of QMEAN was 0.55.PbWRKY had the highest homology with AtWRKY4 and was most likely a homologous protein.

PbWRKY Protein Interaction Network Prediction
To understand the potential regulatory network between PbWRKY proteins, a predicted protein interaction network consisting of 26 PbWRKY proteins was constructed from 60 PbWRKYs using STRING based on homologs of A. thaliana (Figure 7).Among the twenty-six PbWRKY proteins, three genes pertained to Group I, 19 genes pertained to Group II, and four genes pertained to Group III.The 26 nodes were divided into two categories, one of which had only four WRKY proteins that may be collectively associated with a simple regulatory pathway.AtWRKY40 (PbWRKY9, PbWRKY46, and PbWRKY52) was located at the center of the regulatory network and had the highest degree of connectivity.It interacted with AtWRKY18 (PbWRKY47) and AtWRKY60 (PbWRKY44), which together regulate ABA signaling.In addition, the interactions of AtWRKY22 (PbWRKY16 and PbWRKY59) and AtWRKY33 (PbWRKY34 and PbWRKY35) are associated with hypoxic adaptation to waterlogging stress.AtWRKY53 (PbWRKY8 and PbWRKY23) plays a significant role in leaf senescence and is also central to stress responses.AtWRKY70 (PbWRKY42) negatively regulates drought resistance by repressing the expression of drought-inducible genes.The homologs of these seven AtWRKY counterparts, PbWRKY, are also involved in a network of other protein interactions with different AtWRKY functions.In summary, these results indicate that there are multiple interactions between PbWRKY proteins in response to multiple stresses.

PbWRKY Protein Interaction Network Prediction
To understand the potential regulatory network between PbWRKY proteins, a predicted protein interaction network consisting of 26 PbWRKY proteins was constructed from 60 PbWRKYs using STRING based on homologs of A. thaliana (Figure 7).Among the twenty-six PbWRKY proteins, three genes pertained to Group I, 19 genes pertained to Group II, and four genes pertained to Group III.The 26 nodes were divided into two categories, one of which had only four WRKY proteins that may be collectively associated with a simple regulatory pathway.AtWRKY40 (PbWRKY9, PbWRKY46, and PbWRKY52) was located at the center of the regulatory network and had the highest degree of connectivity.It interacted with AtWRKY18 (PbWRKY47) and AtWRKY60 (PbWRKY44), which together regulate ABA signaling.In addition, the interactions of AtWRKY22

PbWRKY Protein Interaction Network Prediction
To understand the potential regulatory network between PbWRKY proteins, a predicted protein interaction network consisting of 26 PbWRKY proteins was constructed from 60 PbWRKYs using STRING based on homologs of A. thaliana (Figure 7).Among the twenty-six PbWRKY proteins, three genes pertained to Group I, 19 genes pertained to Group II, and four genes pertained to Group III.The 26 nodes were divided into two categories, one of which had only four WRKY proteins that may be collectively associated with a simple regulatory pathway.AtWRKY40 (PbWRKY9, PbWRKY46, and PbWRKY52) was located at the center of the regulatory network and had the highest degree of connectivity.It interacted with AtWRKY18 (PbWRKY47) and AtWRKY60 (PbWRKY44), which together regulate ABA signaling.In addition, the interactions of AtWRKY22 (PbWRKY16 and PbWRKY59) and AtWRKY33 (PbWRKY34 and PbWRKY35) are associated with hypoxic adaptation to waterlogging stress.AtWRKY53 (PbWRKY8 and PbWRKY23) plays a significant role in leaf senescence and is also central to stress responses.AtWRKY70 (PbWRKY42) negatively regulates drought resistance by repressing the expression of drought-inducible genes.The homologs of these seven AtWRKY counterparts, PbWRKY, are also involved in a network of other protein interactions with different AtWRKY functions.In summary, these results indicate that there are multiple interactions between PbWRKY proteins in response to multiple stresses.Protein nodes that are enlarged indicate the availability of three-dimensional protein structure information.Gray lines connect proteins that are associated with recurring text-mining evidence.Line thickness indicates the strength of data support.

Expression Analysis of the PbWRKYs
Waterlogging and drought stress affect plant growth, and the response to biotic and abiotic stresses occurs mainly at the transcriptional level.To determine whether PbWRKYs play a critical role in waterlogging and drought stress responses, we used transcriptome data from two periods (7 and 14 d) under waterlogging stress and three periods (3, 6, and 9 d) under drought stress, as well as rehydration for 3 d after 9 d drought stress, to explore the expression of 60 PbWRKYs under waterlogging and drought stress (Figures 8 and 9).
Under drought stress, most PbWRKY expression patterns could be classified as either low expression in almost all periods, consistently high expression, or high expression at different periods.Drought treatment for 3, 6, and 9 d, followed by 3 d of rehydration, resulted in 35 (58%), 21 (35%), 24 (40%), and 40 (67%) genes being upregulated compared to the control.PbWRKY26 and PbWRKY60 reached the highest expression levels at 3 d with downregulated expression in other periods, and PbWRKY20 had the highest expression at 9 d.PbWRKY10, PbWRKY22, PbWRKY32, PbWRKY50, PbWRKY51, and PbWRKY58 had high expression levels in all periods, while PbWRKY2, PbWRKY4, PbWRKY15, PbWRKY18, PbWRKY21, PbWRKY29, PbWRKY36, PbWRKY38, and PbWRKY53 were expressed at low levels in most periods.Protein nodes that are enlarged indicate the availability of three-dimensional protein structure information.Gray lines connect proteins that are associated with recurring text-mining evidence.Line thickness indicates the strength of data support.

Expression Analysis of the PbWRKYs
Waterlogging and drought stress affect plant growth, and the response to biotic and abiotic stresses occurs mainly at the transcriptional level.To determine whether PbWRKYs play a critical role in waterlogging and drought stress responses, we used transcriptome data from two periods (7 and 14 d) under waterlogging stress and three periods (3, 6, and 9 d) under drought stress, as well as rehydration for 3 d after 9 d drought stress, to explore the expression of 60 PbWRKYs under waterlogging and drought stress (Figures 8 and 9).Real-time quantitative PCR (RT-qPCR) expression levels of selected PbWRKY genes following drought stress and waterlogging stress.PbWRKY gene expression levels were analyzed by RT-qPCR, and RG6, RG7, and RG8 were used as internal reference genes.The relative expression levels were calculated using the 2 −ΔΔCT method.Data represent the mean ± standard deviation (p < 0.05).To calculate the letters, we use least significant difference (LSD).The error bars a-f represent standard error in figures.
The response of PbWRKYs under waterlogging stress was not as pronounced as the response to drought stress, with most genes showing a downregulated expression pattern.Their expression profiles under waterlogging stress could be divided into two categories: low expression in most periods and high expression in different periods.PbWRKY18 and PbWRKY53 reached their highest expression at 7 d and were subsequently downregulated, while PbWRKY4, PbWRKY49, and PbWRKY56 reached their highest expression at 14 d.PbWRKY2, PbWRKY15, PbWRKY21, PbWRKY29, PbWRKY36, and PbWRKY38 were consistently highly expressed, which was different from drought stress (Figure 8).This suggests that PbWRKY expression patterns differed under waterlogging stress.PbWRKY10, PbWRKY20, PbWRKY22, PbWRKY26, PbWRKY32, PbWRKY50, PbWRKY51, PbWRKY58, and PbWRKY60 primarily promoted the P. bournei response under drought stress, whereas PbWRKY2 and PbWRKY4 mainly promoted the P. bournei response under waterlogging stress.Real-time quantitative PCR (RT-qPCR) expression levels of selected PbWRKY genes following drought stress and waterlogging stress.PbWRKY gene expression levels were analyzed by RT-qPCR, and RG6, RG7, and RG8 were used as internal reference genes.The relative expression levels were calculated using the 2 −∆∆CT method.Data represent the mean ± standard deviation (p < 0.05).To calculate the letters, we use least significant difference (LSD).The error bars a-f represent standard error in figures.

Verification of the Expression of PbWRKYs by RT-qPCR
The response of PbWRKYs under waterlogging stress was not as pronounced as the response to drought stress, with most genes showing a downregulated expression pattern.Their expression profiles under waterlogging stress could be divided into two categories: low expression in most periods and high expression in different periods.PbWRKY18 and PbWRKY53 reached their highest expression at 7 d and were subsequently downregulated, while PbWRKY4, PbWRKY49, and PbWRKY56 reached their highest expression at 14 d.PbWRKY2, PbWRKY15, PbWRKY21, PbWRKY29, PbWRKY36, and PbWRKY38 were consistently highly expressed, which was different from drought stress (Figure 8).This suggests that PbWRKY expression patterns differed under waterlogging stress.PbWRKY10, PbWRKY20, PbWRKY22, PbWRKY26, PbWRKY32, PbWRKY50, PbWRKY51, PbWRKY58, and PbWRKY60 primarily promoted the P. bournei response under drought stress, whereas Pb-WRKY2 and PbWRKY4 mainly promoted the P. bournei response under waterlogging stress.

Verification of the Expression of PbWRKYs by RT-qPCR
The expression patterns of 25 selected PbWRKYs under drought and waterlogging stresses were examined by RT-qPCR (Figures 10 and 11).Most genes had significant expression after drought and waterlogging stress treatments.Under drought stress, most genes repressed expression in the D2 and D3 periods and upregulated expression in the D4 period (3 d with water after 9 d of drought stress); however, PbWRKY20, PbWRKY50, PbWRKY55, and PbWRKY57 were upregulated in the D2 and D3 periods; PbWRKY18, PbWRKY36, and PbWRKY38 were upregulated in the D2 period and repressed in the D3 period; and PbWRKY4 repressed expression in the D2 period and regulated expression in the D3 period.The genes with low expression in the D3 period were PbWRKY23, PbWRKY34, and PbWRKY43, and the genes with high expression in the D4 period were PbWRKY23, PbWRKY34, PbWRKY35, PbWRKY43, PbWRKY46, and PbWRKY5.The expression patterns of 25 selected PbWRKYs under drought and waterlogging stresses were examined by RT-qPCR (Figures 10 and 11).Most genes had significant expression after drought and waterlogging stress treatments.Under drought stress, most genes repressed expression in the D2 and D3 periods and upregulated expression in the D4 period (3 d with water after 9 d of drought stress); however, PbWRKY20, PbWRKY50, PbWRKY55, and PbWRKY57 were upregulated in the D2 and D3 periods; PbWRKY18, PbWRKY36, and PbWRKY38 were upregulated in the D2 period and repressed in the D3 period; and PbWRKY4 repressed expression in the D2 period and regulated expression in the D3 period.The genes with low expression in the D3 period were PbWRKY23, PbWRKY34, and PbWRKY43, and the genes with high expression in the D4 period were PbWRKY23, PbWRKY34, PbWRKY35, PbWRKY43, PbWRKY46, and PbWRKY5.same as the CK in the W1 and W2 periods.The gene with the highest expression during the W1 period was PbWRKY36; the genes with relatively high expression during the W1 period were PbWRKY11, PbWRKY18, PbWRKY34, PbWRKY36, PbWRKY38, and PbWRKY43.The gene with the highest expression during the W2 period was PbWRKY43; the genes with relatively high expression during the W2 period were PbWRKY4, PbWRKY34, PbWRKY36, PbWRKY38, and PbWRKY43.In summary, our investigation focused on PbWRKY23, PbWRKY34, and PbWRKY43, specifically observing their responses under drought stress.Similarly, PbWRKY34, PbWRKY36, PbWRKY38, and PbWRKY43 were the focal points of our examination under waterlogging stress conditions.Notably, PbWRKY34 and PbWRKY43 demonstrated distinctive expression patterns, showing specificity under either drought or waterlogging stress, making them particularly intriguing subjects for focused research in these respective environmental conditions.Some genes were upregulated under waterlogging stress, while others were repressed.PbWRKY18, PbWRKY36, PbWRKY38, PbWRKY4, PbWRKY53, and PbWRKY57 were upregulated in the W1 and W2 periods.PbWRKY11, PbWRKY14, PbWRKY16, PbWRKY22, PbWRKY23, PbWRKY27, PbWRKY31, PbWRKY32, PbWRKY34, PbWRKY35, PbWRKY40, PbWRKY42, PbWRKY43, PbWRKY46, PbWRKY5, PbWRKY50, PbWRKY55, and PbWRKY8 were all repressed in the W1 and W2 periods.PbWRKY20 was almost the same as the CK in the W1 and W2 periods.The gene with the highest expression during the W1 period was PbWRKY36; the genes with relatively high expression during the W1 period were Pb-WRKY11, PbWRKY18, PbWRKY34, PbWRKY36, PbWRKY38, and PbWRKY43.The gene with the highest expression during the W2 period was PbWRKY43; the genes with relatively high expression during the W2 period were PbWRKY4, PbWRKY34, PbWRKY36, PbWRKY38, and PbWRKY43.

Discussion
In summary, our investigation focused on PbWRKY23, PbWRKY34, and PbWRKY43, specifically observing their responses under drought stress.Similarly, PbWRKY34, Pb-WRKY36, PbWRKY38, and PbWRKY43 were the focal points of our examination under waterlogging stress conditions.Notably, PbWRKY34 and PbWRKY43 demonstrated dis-tinctive expression patterns, showing specificity under either drought or waterlogging stress, making them particularly intriguing subjects for focused research in these respective environmental conditions.

Construction of Phylogenetic Tree and Multiple Sequence Alignment of PbWRKYs
According to the gene structure and conserved motif analysis, PbWRKYs could be classified into three groups, namely, Groups I, II, and III, among which Group II could be further divided into five subgroups (IIa-IIe).The numbers of the 60 PbWRKYs differed among the different groups.Groups I, II, and III contained thirteen, forty, and seven proteins, respectively.The number of WRKY groups was different in different tree species, such as A. thaliana [17] and Populus tomentosa [25].The number of WRKY groups was largest in Group I, while the number of WRKY groups in O. sativa was previously reported to be largest in Group III [12].However, the number of WRKY groups in P. bournei has been reported to be largest in Group II, which is the same in Eucommia ulmoides, O. fragrans, and Solanum lycopersicum [21,26,27], indicating that more gene duplications have occurred in Group II during evolution.As shown in Figure 2, the PbWRKY proteins of Groups IIa and IIb, as well as IId and IIe, were located close to each other.Therefore, Group II could be divided into five subgroups according to the phylogenetic relationship: IIa, IIb, IIc, IId, and IIe.
The conserved motifs of PbWRKY proteins were then evaluated.As shown in Figure 4, the WRKYGQK motif was highly conserved in PbWRKY proteins, but slight variations were found in four genes (PbWRKY19, PbWRKY29, PbWRKY39, and PbWRKY44).The WRKY motif of PbWRKY19 was replaced by WKKY, the same sequence variation occurred in PbWRKY44, and the domains of PbWRKY29 and PbWRKY39 in Group IIc had deletions.It was previously found that variants of the WRKYGQK motif may affect the selection and regulation of the WRKY TF target genes [28,29], and therefore, the function and binding specificity of PbWRKY19, PbWRKY29, PbWRKY39, and PbWRKY44 are worthy of further research.
The variation in the structural domain is the divergent force of the expansion and evolution of the PbWRKY gene family.Variations in the WRKY domain are common in many species, such as E. ulmoides and Cinnamomum camphora [27,30].There were large variations in the N-terminal domain of WRKY, which could affect its binding activity to DNA targets.However, the PbWRKY19 protein in Group I in P. bournei displayed a domain loss event, and it was located close to the N-terminus.PbWRKY29 and PbWRKY39 in Group IIc and PbWRKY44 in Group III also had domain loss events.WRKYGQK at the conserved site of PbWRKYs became WKKYGQK, and it has been speculated that this variation may produce some new biological functions [31,32].

Protein Conserved Motifs and Gene Structure
According to the protein motif composition, PbWRKYs were relatively well conserved, with multiple conserved motifs.Motifs 1, 2, 5, 8, 14, and 16 appeared in pairs, indicating that they were functionally related to subgroup classification; motifs 14 and motif 17 were distributed irregularly across the seven subgroups.In general, members of the same subgroup shared common motifs.In terms of gene structure, all members of Group IId contained three exons, and the gene structure was conserved; however, different groups had different numbers of exons.For example, most members of Group I had seven exons, and members of Group III had a maximum of three exons, and therefore, the gene structure was relatively simple.The members of Group II had a maximum of seven to eight exons, and therefore, the gene structure was more complex than that of Groups I and III.This distribution of quantities is similar to that of grape and wheat [10,33].

Ka and Ks Evolutionary Selection
Gene duplication events are one of the reasons why different species have different numbers of WRKY gene families.Gene duplication leads to the evolution of WRKY genes, which may result in new functions, thus improving plant stress resistance [34,35].According to the duplication sequences of PbWRKY, the Ka/Ks mutation ratios of 21 pairs of PbWRKY duplicated genes were calculated (Table 2).This ratio is an important indicator of study selection limitations and can be used to estimate the approximate timing of duplication events.The Ka/Ks ratio of 21 pairs of PbWRKYs was <1, which means that the PbWRKYs eliminated deleterious mutation sites through purification selection during evolution and that PbWRKYs were highly conserved.Additionally, a total of six duplication events were found in 60 PbWRKYs and 66 AtWRKYs (Table 3), of which five pairs of Ka/Ks ratios were <1, and only PbWRKY37 and AtWRKY34 had Ka/Ks ratios >1, indicating positive selection between these two genes [36,37].

Protein Tertiary Structure Prediction and Protein Regulatory Networks
The protein regulatory network of the PbWRKY gene family was investigated.In the network, the colored spheres (protein nodes) were used as optical aids to represent the various input proteins and the forecasted interactions.The protein nodes demonstrated the accuracy of the protein three-dimensional model information.Gray lines connect the proteins, which are related to the circular text evidence.The strength of the data support was indicated by the thickness of the line [38,39].AtWRKY40 (PbWRKY9, PbWRKY46, and PbWRKY52) was at the center of the regulatory network and had the most sides, interacting with AtWRKY18 (PbWRKY47) and AtWRKY60 (PbWRKY44), which together regulate ABA signaling [40,41].Additionally, the interactions of AtWRKY22 (PbWRKY16 and PbWRKY59) and AtWRKY33 (PbWRKY34 and PbWRKY35) are associated with hypoxic adaptation to waterlogging stress.AtWRKY53 (PbWRKY8 and PbWRKY23) regulates leaf senescence and is essential for responding to abiotic stresses such as drought [11].At the same time, AtWRKY70 (PbWRKY42) negatively regulates drought resistance by repressing the expression of drought-inducible genes [42], and the interactions of PbWRKY16, PbWRKY59, PbWRKY34, PbWRKY35, PbWRKY8, and PbWRKY23 may be involved in the mechanism of the waterlogging stress response.There were no obvious interactions between these proteins, and a further correlation analysis may be needed to determine if any exist.

Promoter Cis-Acting Elements
Cis-acting elements in the promoter region affect gene expression by binding to TFs [43].The analysis results indicated the number of light response elements in the PbWRKY promoter, followed by hormone response elements and then stress response elements.A total of 60 PbWRKY genes contained 111 G-boxes, which were related to plant hypoxia.They also included hundreds of ABRE response elements and 84 ARE response elements, which were related to anaerobic induction.Finally, they also contained 30 MBS response elements, which are related to the resistance of plants to drought stress.These may respond to drought and waterlogging stress.This discovery suggests that PbWRKYs play an important role in the adaptation of P. bournei to environmental deterioration, such as waterlogging and drought.

The Expression Pattern of PbWRKYs
The expression pattern of PbWRKYs under waterlogging and drought stress was analyzed through the use of heatmaps.An RT-qPCR method was used to verify 25 genes, but only PbWRKY4, PbWRKY18, PbWRKY20, PbWRKY22, PbWRKY23, PbWRKY36, Pb-WRKY38, PbWRKY40, PbWRKY50, PbWRKY53, PbWRKY55, and PbWRKY57 had significant expressions under drought and waterlogging stresses.
The six genes PbWRKY18, PbWRKY36, PbWRKY38, PbWRKY4, PbWRKY53, and Pb-WRKY57 displayed a gradually increasing expression trend under the 7 d waterlogging stress, with the highest expression under the 14 d waterlogging stress.The highest expression was found for PbWRKY36.These six genes were the most sensitive to waterlogging treatments.PbWRKY18, PbWRKY20, PbWRKY22, PbWRKY23, PbWRKY38, PbWRKY40, Pb-WRKY50, PbWRKY55, and PbWRKY57 may play a major role in drought stress.PbWRKY18, PbWRKY36, PbWRKY38, PbWRKY4, PbWRKY53, and PbWRKY57 played a significant role in the waterlogging treatment, while PbWRKY18, PbWRKY38, and PbWRKY57 played positive regulatory roles under both the waterlogging and drought treatments.
The protein interaction network displayed the same trend as in the transcriptome data.PbWRKY46, PbWRKY16, PbWRKY34, PbWRKY35, PbWRKY8, and PbWRKY42 decreased significantly under both drought and waterlogging stress.PbWRKY23 increased first and then decreased under drought stress and decreased significantly under waterlogging stress.

Significantly Expressed PbWRKYs under Drought and Waterlogging Stresses
During drought stress in P. bournei, PbWRKY55 expression was upregulated during D1, D2, and D3, and therefore, it may play a major role in drought stress in P. bournei.AtWRKY61 was located on the same branch as PbWRKY55, with both belonging to Group IIb.Jiang showed that AtWRKY61 expression was significantly increased after drought, cold, and ABSA, which was consistent with the PbWRKY transcriptome.AtWRKY61 may respond to nonbiological stress by interacting with AtWRKY9 [24,44], and PbWRKY55 was in the same position as AtWRKY9 in the protein interaction network.We, therefore, speculated that PbWRKY55 is an important drought-regulating gene in P. bournei.
PbWRKY36 was consistently upregulated and expressed at high values in W1 and W2 during waterlogging stress in P. bournei.We, therefore, speculated that PbWRKY36 plays a significant role in waterlogging stress in P. bournei.AtWRKY4 is located on the same branch as PbWRKY36, and both belong to Group I. Most PbWRKYs were similar to AtWRKY4 in the predicted tertiary structure model of P. bournei proteins.Previous research has shown that AtWRKY4 is a vital salt stress gene; Li and Peng showed that AtWRKY4 was overexpressed under salt and Me-JA stresses.The role of the AtWRKY3 and AtWRKY4 genes in plant defense responses to necrotrophic pathogens has been reported [45].Roots gradually decay and become necrotic under waterlogged conditions, and we, therefore, speculated that PbWRKY36 promotes defense responses through the upregulation of root necrosis under waterlogging stress.
PbWRKY18, PbWRKY38, and PbWRKY57 had positive regulatory roles under both drought and waterlogging stresses.AtWRKY45 is homologous to PbWRKY18, and previous studies have reported that it displays a strong response to jasmonate treatment [46].AtWRKY45 also responds strongly under low-phosphorus stress.AtWRKY6 is homologous to PbWRKY38, and the expression of AtWRKY6 is affected by the triggering of senescence and plant defense response signals.AtWRKY6 overexpression has been observed in the presence of low levels of phosphorus, and eight potential target genes have been identified, including 14-3-3 protein, tryptophan synthase, carbonic anhydrases, ATP synthase, and Gln synthetase [47].AtWRKY15 is homologous to PbWRKY57 and regulates plant growth and salt stress responses [48].CsWRKY7, AtWRKY7, and AtWRKY15 are homologous genes.CsWRKY7 is localized to the nucleus and is involved in the plant response to environmental stress and growth [49].Therefore, we hypothesized that PbWRKY18, PbWRKY38, and PbWRKY57 are all important defense genes in P. bournei.

Different Transcription Factors under Drought and Waterlogging Stresses
Transcription factors can regulate the expression of multiple stress-related genes to improve plant stress resistance.Therefore, by overexpressing plant-specific transcription factors in plants, they can simultaneously regulate multiple functional genes and thus improve plant stress resistance, which has become a research focus in genetic improvement and breeding of plant stress resistance.WRKY, MYB, and AP2/ERF are the main transcription factors related to drought and flood resistance in plants.
The WRKY gene family is one of the larger gene families in plants, and the WRKY gene can specifically bind to the downstream gene promoter cis-acting element, which contains a very conserved (C/T) TGAC (T/C) sequence, so it is called W-box.The WRKY gene binds to this element, thereby activating or inhibiting the expression of downstream genes.Drought stress and waterlogging are the most common abiotic stress factors suffered by plants.Plants can regulate their resistance to stress through the WRKY gene regulatory network and hormone signal transduction.The overexpression of the GhWRKY6-like gene in cotton can enhance the tolerance of cotton to drought and osmotic stress, while the MdWRKY30 gene can enhance the tolerance of overexpressed apple callus to salt and osmotic stress by regulating the expression of downstream related genes.It was also found that Arabidopsis thaliana had stronger salt tolerance and osmotic ability than the wild type.In addition, the WRKY gene can also regulate the content of osmotic regulatory substances, such as soluble sugar and proline, and reduce the content of malondialdehyde so as to improve the drought resistance of plants.
MYB is one of the largest transcription factor families in plants.All genes in this family contain a specific conserved domain.The MYB domain consists of 1-4 tandem and non-repetitive R motifs.Numerous studies have shown that MYB genes are involved in abiotic stress pathways and resist stress through their transcriptional regulatory networks.MYB gene is also directly involved in the regulation of external environmental signals.MYB plays an important role in drought stress, and PtoMYB170 plays a role in the regulation of secondary wall synthesis.Meanwhile, drought experiments in Arabidopsis Thaliana showed that the PtoMYB170 gene plays a regulatory role in drought stress.In addition, it was found that the MYB156 and MYB221 genes of poplar, the AtMYB14 gene of Arabidopsis Thaliana, the MdMYB121 gene of apple, and the ApMYBs gene of Andrographis andrographis were involved in the regulation of drought stress.
AP2/ERF gene is a transcription factor unique to plants, and its family genes are numerous and versatile.According to the sequence similarity and the number of AP2/ERF functional domains, the AP2/ERF gene family can be divided into AP2, RAV, ERF, and Soloist subfamilies, and the ERF subfamily can be further divided into DREB and ERF subfamilies.DREB gene regulates the expression of related genes by binding to DRE elements in the downstream gene promoter region, the ERF gene activates downstream gene expression by binding to GCC-box elements in the downstream target gene promoter region, and RAV and ERF subfamily genes participate in plant hormone response and abiotic stress response.At present, a large number of AP2/ERF genes have been identified in Arabidopsis Thaliana and rice, and gene function analysis has found that AP2/ERF genes are involved in abiotic stress responses such as waterlogging and drought.BpERF13 overexpressed transgenic strains of Betulae alba were subjected to cold stress, and it was found that SOD, POD, and CBF genes of transgenic strains were significantly upregulated, indicating that transgenic strains were more cold-resistant.The upregulated expression of AcERF74 in kiwifruit is involved in the waterlogging response of kiwifruit, and the upregulated expression of ZmEREB102 in maize is involved in the drought resistance response of maize.In summary, AP2/ERF gene family members are widely involved in abiotic stress responses such as waterlogging, salt, and drought.

Multiple Sequence Alignment and Phylogenetic Analysis
The phylogenetic relationship among the aligned sequences obtained was estimated by the neighbor-joining (NJ) method using the MEGA 7.0 software [52].Further, phylogenetic tree beautification was performed by Evolview 8 (https://www.evolgenius.info/evolview(accessed on 7 June 2023)).Ultimately, the protein evolutionary tree consisted of the WRKY family members of A. thaliana and P. bournei.

Calculation of Non-Synonymous (Ka) and Synonymous (Ks) Values
Non-synonymous and synonymous substitution events were estimated using Ka/Ks values, and Ks and Ka replacement rates were calculated using the Nei and Gojobori methods implemented in the KaKs calculator (http://code.google.com/p/kaks-calculator/wiki/KaKs_Calculator (accessed on 15 June 2023)) [55].

Protein Tertiary Structure Prediction and Interaction Network Analysis
The protein tertiary structure was predicted using SWISS-MDEL (https://swissmodel .expasy.org/interactive (accessed on 27 June 2023)), and the highest percentage of protein tertiary structure models was found for each group.Using Ortho Venn 2 (https://orthoven n2.bioinfotoolkits.net/home(accessed on 28 June 2023)) and String (https://string-db.org/(accessed on 29 June 2023)), the WRKY protein sequences of the two species were compared, and protein interaction networks were mapped.

Plant Materials and Abiotic Stress Treatments
Three hundred healthy P. bournei seedlings (three years old) were obtained from the Fujian Academy of Forestry Sciences and then transplanted to the Fujian Agriculture and Forestry University nursery for drought and waterlogging stress experiments.Place 300 young seedlings in a greenhouse, and for the seedlings under drought stress, no water is provided directly.For the seedlings under waterlogging stress, a large pot is placed inside a smaller pot so that the roots of the seedlings are fully submerged in water.Drought and waterlogging stress occurred between 29 July and 25 August 2021.Prior to 29 July, we initiated several drought stress experiments.However, due to weather conditions, i.e., the start of a rain event after 3 d of drought, we had to terminate the experiment.There was no rain from 29 July to 10 August.According to previous experiments, the seedlings were inactivated for 12 d under drought and 35 d under waterlogging stress.We took photographs and root samples after treatments of 0, 3, 6, and 9 d under drought stress and 3 d after rewatering.The post-stress recovery was suitable for drought-affected plants.During the pre-experiment period, we conducted recovery experiments on plants under waterlogging stress for 28 d, but at this time, the seedlings had already become inactive and could not be measured for transcriptome and physiological indicators.We took photographs and root samples after waterlogging treatments of 0, 7, 14, 21, and 28 d.Each sampling used the same procedure and was repeated five times, and the whole plant, as well as its roots and leaves, were photographed with a Leica M Type 240 camera (Figures S1 and S2).Generally, mature trees have better tolerance to environmental stress.Field observations have shown that seedlings are more likely to die when drought or waterlogging occurs.Because this was a controllable stress test, small seedlings were easy to work with.Currently, only the response characteristics of the seedling stage to stress are understood, and there are no clear results for the study of older trees.

RT-qPCR Analysis
Each sample was weighed to 0.1−0.2g and ground rapidly in liquid nitrogen.The total RNA of P. bournei was extracted from different periods using a TIANGEN RNA prep Pure (DP441) Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (centrifuge column), which was designed to prevent RNase contamination.The integrity of the RNA was checked using 1% agarose gel electrophoresis, and the concentration and purity were measured by ultraviolet spectroscopy.RNA was used for cDNA synthesis.
cDNA was synthesized using the NovaBio HyperScript TM III RT Super Mix for RT-qPCR with the gDNA Remover Kit.Genomic DNA was removed to constitute a 20 µL reaction system (Table S1).RG6, RG7, and RG8 were used as internal reference genes [56].The RT-qPCR technique was used to examine the expression of 11 internal reference genes at seven different times in the roots of P. bournei.The NovaBio 2*S6 Universal SYBR RT-qPCR Mix kit (operated on an Icycler iQ5 (BioRad, Hercules, CA, USA)) was used for this analysis.The total reaction system was 20 µL in a 96-well plate (Table S2), and three repeated operations were performed for each sample.

Conclusions
We studied the evolution of the WRKY gene family in P. bournei and other species and systematically identified the PbWRKY gene family in P. bournei at the genome-wide level.A total of 60 PbWRKY genes were identified and classified into three groups.These genes had similarities and differences in physicochemical properties, gene structure, conserved motifs, cis-regulatory elements, gene duplication, and the protein interaction network.Transcriptome data indicated that PbWRKY4, PbWRKY18, PbWRKY20, PbWRKY22, PbWRKY23, PbWRKY36, PbWRKY38, PbWRKY40, PbWRKY50, PbWRKY53, PbWRKY55, and PbWRKY57 had significant expression under drought and waterlogging stresses.The results serve not only as a foundation for the selection of alternative genes for abiotic stress responses but will also inform future studies of the effects of WRKY in woody plants.

Figure 1 .
Figure 1.Phylogenetic tree of WRKY proteins from P. bournei and Arabidopsis thaliana.The tree was divided into seven phylogenetic groups distinguished by color.The colors yellow, red, green, blue, orange, purple, and pink represent Groups I, IIa, IIb, IIc, IId, IIe, and III, respectively.The names of the subfamilies are shown outside of the circle.

Figure 1 .
Figure 1.Phylogenetic tree of WRKY proteins from P. bournei and Arabidopsis thaliana.The tree was divided into seven phylogenetic groups distinguished by color.The colors yellow, red, green, blue, orange, purple, and pink represent Groups I, IIa, IIb, IIc, IId, IIe, and III, respectively.The names of the subfamilies are shown outside of the circle.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 26WRKYGQK domain and a CX7CX23HXC zinc-finger structure were found in Group III PbWRKYs.

Figure 2 .
Figure 2. Multiple sequence alignment of the WRKY domains of 60 PbWRKY proteins.I-N, I-C, IIa, II-b, II-c, II-d, II-e, and III represent different subgroups in three subfamilies; colors indicate sequence similarity; and a darker color indicates higher conservatism.

Figure 2 .
Figure 2. Multiple sequence alignment of the WRKY domains of 60 PbWRKY proteins.I-N, I-C, II-a, II-b, II-c, II-d, II-e, and III represent different subgroups in three subfamilies; colors indicate sequence similarity; and a darker color indicates higher conservatism.

Figure 4 .
Figure 4. Prediction of cis-regulatory elements in the promoter regions of the PbWRKY gene family.Figure 4. Prediction of cis-regulatory elements in the promoter regions of the PbWRKY gene family.

Figure 4 .
Figure 4. Prediction of cis-regulatory elements in the promoter regions of the PbWRKY gene family.Figure 4. Prediction of cis-regulatory elements in the promoter regions of the PbWRKY gene family.

Figure 5 .
Figure 5. Cis-element analysis in the promoter of PbWRKY genes.The cis-element distributed in the promoters of PbWRKY.The number of cis-acting elements in the promoter region of each PbWRKY gene.

Figure 5 .
Figure 5. Cis-element analysis in the promoter of PbWRKY genes.The cis-element distributed in the promoters of PbWRKY.The number of cis-acting elements in the promoter region of each PbWRKY gene.

Figure 6 .
Figure 6.Prediction of protein tertiary structure.(A−G) represent Groups I, IIa, IIb, IIc, IId, IIe, and III, respectively, and represent the most important protein structure predictions.

Figure 7 .
Figure 7. Regulatory networks of the PbWRKY gene family.Colored balls (protein nodes) in the network were used as a visual aid to indicate different input proteins and predicted interactions.

Figure 6 .
Figure 6.Prediction of protein tertiary structure.(A-G) represent Groups I, IIa, IIb, IIc, IId, IIe, and III, respectively, and represent the most important protein structure predictions.

26 Figure 6 .
Figure 6.Prediction of protein tertiary structure.(A−G) represent Groups I, IIa, IIb, IIc, IId, IIe, and III, respectively, and represent the most important protein structure predictions.

Figure 7 .
Figure 7. Regulatory networks of the PbWRKY gene family.Colored balls (protein nodes) in the network were used as a visual aid to indicate different input proteins and predicted interactions.

Figure 7 .
Figure 7. Regulatory networks of the PbWRKY gene family.Colored balls (protein nodes) in the network were used as a visual aid to indicate different input proteins and predicted interactions.

Figure 8 .
Figure 8. Heatmaps of expression profiles of PbWRKY genes in response to drought and waterlogging.CK represents normal watering; W1 represents 7 d under waterlogging stress; W2 represents 14 d under waterlogging stress; D1 represents 3 d under drought stress; D2 represents 6 d under drought stress; D3 represents 9 d under drought stress; D4 represents 3 d with water after 9 d of drought stress.Under drought stress, most PbWRKY expression patterns could be classified as either low expression in almost all periods, consistently high expression, or high expression at different periods.Drought treatment for 3, 6, and 9 d, followed by 3 d of rehydration, resulted in 35 (58%), 21 (35%), 24 (40%), and 40 (67%) genes being upregulated compared to the control.PbWRKY26 and PbWRKY60 reached the highest expression levels at 3 d

Figure 8 .
Figure 8. Heatmaps of expression profiles of PbWRKY genes in response to drought and waterlogging.CK represents normal watering; W1 represents 7 d under waterlogging stress; W2 represents 14 d under waterlogging stress; D1 represents 3 d under drought stress; D2 represents 6 d under drought stress; D3 represents 9 d under drought stress; D4 represents 3 d with water after 9 d of drought stress.

Figure 9 .
Figure 9. Real-time quantitative PCR (RT-qPCR) expression levels of selected PbWRKY genes following drought stress and waterlogging stress.PbWRKY gene expression levels were analyzed by RT-qPCR, and RG6, RG7, and RG8 were used as internal reference genes.The relative expression levels were calculated using the 2 −ΔΔCT method.Data represent the mean ± standard deviation (p < 0.05).To calculate the letters, we use least significant difference (LSD).The error bars a-f represent standard error in figures.

Figure 9 .
Figure 9. Real-time quantitative PCR (RT-qPCR) expression levels of selected PbWRKY genes following drought stress and waterlogging stress.PbWRKY gene expression levels were analyzed by RT-qPCR, and RG6, RG7, and RG8 were used as internal reference genes.The relative expression levels were calculated using the 2 −∆∆CT method.Data represent the mean ± standard deviation (p < 0.05).To calculate the letters, we use least significant difference (LSD).The error bars a-f represent standard error in figures.

Figure 10 .Figure 10 .
Figure 10.Expression patterns of 25 PbWRKY genes under drought stress.Gene expression levels at 0, 3, 6, and 9 d under drought stress and at 3 d with water after 9 d of drought stress.

Table 1 .
Details of information on the WRKY transcription factor family in P. bournei.

Table 2 .
Ka/Ks analysis and divergence time estimated for P. bournei duplicated WRKY paralogs.

Table 3 .
Ka/Ks analysis and estimated divergence time of WRKY genes from P. bournei and A. thaliana.

Table 4 .
The number and type of WRKY proteins in 28 plants.